In a flow cytometer, sample particles are passed through a small aperture in a flow cell (sometimes referred to as a measuring chamber). The small aperture confines the particles to an interrogation zone or region where they can then be evaluated.
In a particle analyzer, for example a flow cytometer, particles such as cells arranged in a sample stream pass through one or more excitation light beams with which the particles interact. Light scattered or emitted by the particles upon interaction with the one or more excitation beams is collected, detected, and analyzed to characterize and differentiate the particles. For example, forward scattering of an excitation beam along its axis may provide information about particle size, side scattering of an excitation beam orthogonally to its axis may provide information about particle internal structure or internal complexity, and fluorescence excited by the one or more excitation beams may provide information about the presence or absence in the particles of fluorophores correlating with particular chemical or biological properties of the particles.
The performance of the analyzer is affected by the proper spatial alignment and positioning of the particles in the sample stream as they pass through the interrogation region. For example, it is desirable to have the particles in a linear arrangement so that the particles will pass one-by-one through the interrogation region where the excitation light will impinge on the particles. If two or more particles are introduced into the interrogation region at the same time, an errant measurement may result as the multiple particles may be interpreted as a single particle. Also, it is desirable to have the particles travel in a spatially consistent path so that the focal point of the excitation light may consistently interrogate each particle. Spatial variation in the transverse direction can cause a reduction in the measurement resolution of the analyzer.
Many conventional analyzers attempt to align particles entrained in the sample stream by hydrodynamic focusing. In hydrodynamic focusing, a suspension of particles is injected into the center of a laminar sheath fluid flow. The forces of the sheath fluid confine the sample stream to a narrow core, thereby aligning the particles entrained therein. Although this technique is commonly used, the sample stream rate of conventional flow cytometers is typically between 10-30 μL/min in order to maintain acceptable measurement resolution. A higher stream rate causes the sample stream core to increase thereby increasing the spatial variation of the particles in the transverse direction. The decrease in spatial reproducibility within the interrogation zone results in loss of the analyzer's measurement resolution.
Other attempts to focus the sample stream have been made. One method is to reduce the diameter of the sample injector thereby narrowing the sample stream core before injecting the stream into the middle of the laminar sheath flow. However, one shortcoming of this technique is that the system is prone to clogging the sample injector, which is undesirable. Another existing method to focus the sample stream is to use ultrasonic waves to focus the particles. Such techniques, however, add complexity to the system.
Accordingly, there is a need for an improved flow cell and method to align particles in a flow cell that improves flow stream rate while maintaining measurement resolution. Additional benefit of the improved device and method may be to avoid adding complexity or increasing potential for clogging the system.